Intestinal microbiome and maternal mental health: preventing parental stress and enhancing resilience in mothers

The number of mothers suffering from mental illness is increasing steadily, particularly under conditions of the coronavirus pandemic. The identification of factors that contribute to resilience in mothers is urgently needed to decrease the risks of poor physical and psychological health. We focused on the risk of parenting stress and psychological resilience in healthy mothers with no psychiatric and physical disorders and conducted two studies to examine the relationships between intestinal microbiota, physical condition, and psychological state. Our results showed that alpha diversity and beta diversity of the microbiome are related to high parenting stress risk. Psychological resilience and physical conditions were associated with relative abundances of the genera Blautia, Clostridium, and Eggerthella. This study helps further understand the gut–brain axis mechanisms and supports proposals for enhancing resilience in mothers.

The study utilizes two cohort studies to investigate the relationship between maternal faecal microbiome and parenting stress.The first study utilizes a large cohort study "The Principle of Human Social Brain-Mind Development" with 339 participants.Study 2 utilizes another cohort study Investigation of the Interrelationship among Mental, Physical, and Intestinal Microbiota in Postpartum Mothers and Their Infants.Investigating 27 first time mothers.The authors found significant association between reported parental stress and changes in the alpha and beta diversity of the gut microbiome of the mothers.In particular, the presence of and butyrate-producing bacteria (e.g., Lachnospira).Study 2 also investigated the association of the microbiome composition with autonomic nervous system functon(oxytocin), physical condition (e.g., body composition, physical function, and physical condition), and psychological state (questionaires).
The study methodology appears to be sound and of high scientific quality.The sample collection, microbiome analysis and statistical investigations are sound and of high scientific quality.Both questionaires/surveys used to assess the psychological and physical well being of the mothers appear robust and well validated.Results are appropriately displayed, please see comment below regarding table 1.The discussion is in depth and considered.Care must be given to attributing causation rather than association to the presence of higher relative abundances of particular bacteria, however the assumptions are well supported by the references.Conclusions appropriate.This study is of high quality and adds to the body of literature in the field, it is novel in that it seeks to describe markers of resilience.
1. Line 20 suggest that "functioning" be changed to health 2. Line 30 change "cannot be restrained."to " is likely to continue to increase" 3. Table 1 needs to be re-formatted as it is very busy and difficult to read.Perhaps if the Shannen alpha diversity for each organism is displayed in Figure 2 then this information could be removed from table 1. Lists of organisms are perhaps better for an appendix and visual representation more powerful for a general audience.4. The structure of the discussion should be changed for clarity.Perhaps discussing each of the features as separate paragraphs would improve the readability.For instance rather than referring to feature 1 the paragraph (line 318) could begin, "abnormal levels of short chain fatty acid producing bacteria have been reported by others." 5. Line 305 change "conditions" to "condition" 6. Line 312 change "less" to "lower" 7. Line 338 start sentence with "Study 2 was designed to we focus on primiparous mothers" 8. Line 344 the statement is confusing: "the diversity of the intestinal microbiota could contribute to psychological resilience through individual differences in vagal activity."Consider revising, as it is unclear if the vagal activity is the bacteria or the mother.9. Line 348 "explained the individual differences" should be was associated with as there is no evidence for causation 10.Line 378 consider revising the introduction to "limitations of the study" Reviewer #3 (Remarks to the Author): The authors have presented some intriguing and novel findings connecting features of the gut microbiome with psychological and physical health among two distinct samples of Japanese mothers.Although these findings are just cross-sectional, I believe that they have the potential to lay the groundwork for future work in this area.As a side note, I was shocked by the finding that many of the new mothers in Study 2 had sarcopenia and poor physical functon(e.g., grip strength), and this finding is certainly worthy of further exploration.There are several major and minor points that I hope the authors will address to increase the scientific soundness of this manuscript: Major points: (1)I do not see hypotheses specified anywhere.As a result, many of the results that are reported seem haphazard, as if the authors are just reporting whatever they happened to find that was significant.For example, in study 1, I don't see any findings reported for the correlations between the gut microbiome and physical functioning even though that appears to be a major focus of this article (and Study 2).In fact, physical functioning was not given much focus in the introduction and it appears to come out of the blue in the results section.Please rework the introduction and results section to show a coherent line of inquiry and specific hypotheses.The results section should then mirror these specific hypotheses.
(2) There are no covariates, which is problematic given that many of these associations (e.g., HRV and the microbiome) may be highly confounded by other factors like general health, exercise, etc.
(3)What is "nonclinical" in reference to the mothers?May want to use a more specific term in the abstract.How was it determined that they were free of psychiatric and physical disease?(4)In study 1, how was the age range of 0-4 years old determined?Was this study specifically designed for these analyses, or are these secondary analyses?(5) How was the specific vagal activity metric chosen?How was respiratory rate controlled for?Please provide more specifics about what participants were doing just prior to and during the heart rate recording.Were they seated, lying down, standing up?Were they holding an infant?(6) Many important methodological details are only reported in the supplemental material, but they should be moved to the main text for reproducibility.Also critical details about the gut microbiome methods are not included in this manuscript (just a citation), but they should be included so that this could be a standalone manuscript.
(9) Why were only mothers included and not fathers?(10) It seems strange that only significant findings were only reported in text and null findings are reported in supplemental material.For example, in study 2, the null relationship between the gut microbiome and parenting stress should be reported in the main text given that it is a major focus of this manuscript.Also please comment on why this association might be null in Study 2 but not Study 1. (11) Was the finding of a negative association between oxytocin and resilience hypothesized?(12) Please provide a citation to support the relative within-person stability of the microbiome over the 2-3 day period that you have between the study visit and the stool sample collection.(13) Please specify whether you tested relationships between all taxa in the microbiota or just the SCFA producers.Either way, there were many statistical tests run, but it appears that you only did a multiple test correction in Study 1, which could be problematic.Do any significant results survive in Study 2 with a multiple test correction?
Minor points: (1)Please explain in the introduction why SCFA producers are important in terms of the gut-brain axis.
(2) Line 93 should read alpha diversity rather than just alpha.
(3) In line 110, please provide definitions of acronyms PSI-P and PSI-C.(4) Have any other studies shown high risk of sarcopenia and poor physical functioning in new mothers?
(5) Line 247 should read "partially explained" rather than "explained" (6) The authors mention certain facets of Japanese life (e.g., diet) that might be relevant to the variables of interest.Please comment in the limitations section about how these results may have been influenced by the fact that this was conducted in Japan.(7) Figure 5

Response to Reviewers
We are grateful to the reviewers for their thorough evaluation of our manuscript and providing valuable comments.We have modified the manuscript accordingly.
In the revised manuscript, the changes made are noted in red font color.
We hope you will find the revised manuscript suitable for publication in your esteemed journal.

Reviewer #1:
1.The major finding of this study is that parenting stress in Japanese mothers is associated with their intestinal microbial dysbiosis, their autonomic nervous system, and their physical condition.The authors are claiming that mothers with high parenting stress have significantly reduced microbial diversity, abnormal short-chain fatty acid-producing bacteria, and increased inflammatory microbiota.
2. Overall, it is a well-written manuscript.
3. The study is novel as it was done first time on Japanese mothers.However, it is an observational study and only correlation is established.A longitudinal study with probiotic treatment would help to strengthen the relationship.

Response to 1-3
Thank you for reviewing our manuscript.We recognize that a longitudinal study with probiotic treatment would be preferable to this correlational study.We hope to conduct a longitudinal research on this topic in the future.================ 4. The study is cross-sectional and carried out for a short period of time.The fecal samples are collected only once.For studying intestinal microbiota, it is important to have at least two point samples (at the beginning and end).It is because the microbiota gets changed for several reasons apart from diet and stress.

Response
We acknowledge the importance of multiple sample points for studies of microbiota.However, to meet our specific research objectives, this was intentionally designed as a cross-sectional study that observed and analyzed the microbiota at a single time point.Also, with over 300 participants, our study provides a robust dataset even with a single sample per individual.Additional sampling would have incurred substantial costs and logistical challenges, which we carefully weighed against the benefits during the design phase.We believe our methodology is justified given our research aims and resource considerations.

Response
We thank the reviewer for pointing this out.We have corrected the typo.

Reviewer #2:
The study utilizes two cohort studies to investigate the relationship between maternal faecal significant association between reported parental stress and changes in the alpha and beta diversity of the gut microbiome of the mothers.In particular, the presence of and butyrate-producing bacteria (e.g., Lachnospira).
Study 2 also investigated the association of the microbiome composition with autonomic nervous system functon(oxytocin), physical condition (e.g., body composition, physical function, and physical condition), and psychological state (questionaires).
The study methodology appears to be sound and of high scientific quality.The sample collection, microbiome analysis and statistical investigations are sound and of high scientific quality.Both questionaires/surveys used to assess the psychological and physical well being of the mothers appear robust and well validated.

Results are appropriately displayed, please see comment below regarding table 1.
The discussion is in depth and considered.Care must be given to attributing causation rather than association to the presence of higher relative abundances of particular bacteria, however the assumptions are well supported by the references.

Conclusions appropriate.
This study is of high quality and adds to the body of literature in the field, it is novel in that it seeks to describe markers of resilience.
1. Line 20 suggest that "functioning" be changed to health

Response
Thank you for this advice.We have changed the content accordingly (Lines 29-30).================ 3. Table 1 needs to be re-formatted as it is very busy and difficult to read.Perhaps if the Shannen alpha diversity for each organism is displayed in Figure 2 then this information could be removed from table 1. Lists of organisms are perhaps better for an appendix and visual representation more powerful for a general audience.

Response
Thank you for your feedback.The Shannon alpha diversity information was originally displayed in Figure 2. We agree that the information in Figure 2 and Table 1 overlap.Therefore, we have kept the results for the psychological and physical indices shown in Table 1 in the main text, moved the intestinal microbiota results to the Supplementary Materials, and modified the table to make it more comprehensible.================ 4. The structure of the discussion should be changed for clarity.Perhaps discussing each of the features as separate paragraphs would improve the readability.For instance, rather than referring to feature 1 the paragraph (line 318) could begin, "abnormal levels of short chain fatty acid-producing bacteria have been reported by others."

Response
Thank you for helping us to clarify our line of thought.In the revised manuscript, we have modified the paragraph in question as you have suggested to improve readability as follows.
Lines 363-364: Regarding the first feature (i), abnormal levels of short-chain fatty acid-producing bacteria have been reported by previous a study.Regarding the second and third features (ii and iii), previous research has found associations between psychiatric disorders and immunity and inflammation-related intestinal bacteria.================ 5. Line 305 change "conditions" to "condition." 6. Line 312 change "less" to "lower." 7. Line 338 start sentence with "Study 2 was designed to focus on primiparous mothers."

5-7. Response
We thank the reviewer for this comment.We have made the advised alterations in Lines 350, 357, and 385, respectively.================ 8. Line 344 the statement is confusing: "the diversity of the intestinal microbiota could contribute to psychological resilience through individual differences in vagal activity."Consider revising, as it is unclear if the vagal activity is the bacteria or the mother.

Response
Thank you for your feedback.We have modified this statement for improved clarity as follows.
Lines 393-395:"Assessing individual differences in both gut microbiota and vagal activity may help to clarify the physiological factors that contribute psychological resilience."================ 9. Line 348 "explained the individual differences" should be was associated with as there is no evidence for causation 10.Line 378 consider revising the introduction to "limitations of the study"

9-10. Response
We agree with this suggestion and have altered the text in Lines 395, 429, respectively.

Reviewer #3:
The authors have presented some intriguing and novel findings connecting features of the gut microbiome with psychological and physical health among two distinct samples of Japanese mothers.Although these findings are just cross-sectional, I believe that they have the potential to lay the groundwork for future work in this area.As a side note, I was shocked by the finding that many of the new mothers in Study 2 had sarcopenia and poor physical functon(e.g., grip strength), and this finding is certainly worthy of further exploration.There are several major and minor points that I hope the authors will address to increase the scientific soundness of this manuscript:

Major points
(1)I do not see hypotheses specified anywhere.As a result, many of the results that are reported seem haphazard, as if the authors are just reporting whatever they happened to find that was significant.For example, in study 1, I don't see any findings reported for the correlations between the gut microbiome and physical functioning even though that appears to be a major focus of this article (and Study 2).In fact, physical functioning was not given much focus in the introduction and it appears to come out of the blue in the results section.
Please rework the introduction and results section to show a coherent line of inquiry and specific hypotheses.The results section should then mirror these specific hypotheses.

Response
In the original manuscript, we tried to keep the Introduction simple and concise.However, as this has provided insufficient information, we have made considerable revisions to this section.In particular, per the reviewer's suggestion, we have added to our descriptions of physical functioning, the autonomic nervous system, and relevant hormones.We have also revised the hypothesis to make it more explicit and have attempted to improve the clarity of the Introduction and Results.We have also cited the paper recommended in comment (8).

Response
General health was controlled to some extent because only healthy mothers who were not currently hospitalized due to physical or mental illness were included.In addition, as the cohort consisted only of Japanese mothers of children aged 0-4 years, it is to be expected that our participants would share more sociodemographic characteristics with one another than would a more general adult sample (of mixed gender and diverse age groups).However, we agree that we should have attempted to examine covariates.Therefore, we reanalyzed maternal years of education and maternal age as covariates potentially related to health status and dietary habits, which were valid indexes for covariates among the measured variables.We found the results to be unchanged (Study 1).The results of this analysis are reported in the main text and the Supplementary Materials.For Study 2, we would have liked to incorporate covariates, but due to the small sample size, it is likely that the results would have been statistically invalid with their inclusion.We have explained this in the is very nice.(8) Please define acronyms the first time they are presented in the main text.(9) You may want to use this article to support the fact that you used V1V2 of 16s: Kameoka, S., Motooka, D., Watanabe, S. et al.Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1-V2 and V3-V4 primer sets.BMC Genomics 22, 527 (2021).https://doi.org/10.1186/s12864-021-07746-4 For your reference, here are some other examples of cross-sectional gut microbiome studies with a single sample from each individual.Osakunor, D.N.M., Munk, P., Mduluza, T., et al.The gut microbiome but not the resistome is associated with urogenital schistosomiasis in preschool-aged children.Commun Biol 3, 155 (2020).https://doi.org/10.1038/s42003-020-0859-7Radjabzadeh, D., Bosch, J.A., Uitterlinden, A.G., et al.Gut microbiome-wide association study of depressive symptoms.Nat Commun 13, 7128 (2022).https://doi.org/10.1038/s41467-022-34502-3================ 5.The first study among the two is statistically well-planned.
microbiome and parenting stress.The first study utilizes a large cohort study "The Principle of Human Social Brain-Mind Development" with 339 participants.Study 2 utilizes another cohort study Investigation of the Interrelationship among Mental, Physical, and Intestinal Microbiota in Postpartum Mothers and Their Infants.Investigating 27 first time mothers.The authors found